Mouse memory b cell isolation kit

BALB/c mice were orally vaccinated four times with NCK2310 (10⁹ CFU/mouse), once a week, followed by 2 boosts (Fig. 1A). Seven days later, splenic memory B cells were magnetically sorted using the mouse memory B cell isolation Kit (Miltenyi Biotec, San Diego, CA). Briefly, 50 μL each of Anti-IgG1-APC, Anti-IgG2ab-APC and 100 μL Biotin Antibody Cocktail were incubated with 108 isolated splenic cells from the vaccinated mice resuspended in 300 μL PBS containing 0.5% FBS and 2 mM EDTA for 5 min on ice. 200 μL of Anti-Biotin microbeads were added to the mix to remove non-memory B cells using LD columns. Anti-APC microbeads were introduced in the flow through of LD columns to further purify memory B cells from the cellular suspension using MS columns. The isolated cells were counted to prepare a cellular suspension containing 10⁶ cells/100 μL. Memory B cells (10⁶ cells/mouse) were then intraperitoneally transferred into recipient naïve BALB/c mice. One month later, recipient mice were intoxicated with BoNT/A complex (1μg/mouse). Mouse survival was monitored.